1. Field of the Invention
The present invention relates to a method and reagent for classifying leukocytes in animal blood.
2. Description of the Related Art
In treatment or diagnosis of canine and feline diseases, blood examination is an essential examination. Information useful for treatment or diagnosis of leukemia, infections and allergic diseases is obtained in examination of leukocytes, and information useful for treatment and diagnosis of anemia is obtained in examination of hemoglobin.
The method of measuring human leukocytes has already been established. In the method, a lysing reagent is added to a human blood sample to lyse erythrocytes in the sample, and the remaining leukocytes are counted with a hemocytometer. By devising ingredients of the lysing reagent, it is possible not only to count total leukocytes, but also to classify the leukocytes into a plurality of sub-groups and count leukocytes in a specific sub-group (see, for example, U.S. Pat. Nos. 4,346,018, 4,485,175 and 5,116,539 and Japanese Laid-Open Patent Application Publication Nos. H02-031162 and H03-020667). Alternatively, a plurality of aliquots are prepared from a blood sample, and different lysing reagents are added to the respective aliquots, to measure specific sub-groups of leukocytes, and from the obtained results, the leukocytes can be classified into 5 sub-groups.
In measurement of hemoglobin, a cyan methemoglobin method is used as an international standard method. However, the cyan methemoglobin method makes use of a toxic cyan compound, thus making disposal of waste-fluid necessary. Therefore, in recent years, hemoglobin is also measured by a method of using no cyan compound; for example, SLS hemoglobin method, methemoglobin method etc. (see, for example, U.S. Pat. Nos. 5,250,437 and 5,968,832).
A hemocytometer for animal blood has been commercially available. In the hemocytometer, a reagent for human blood is used, and a program for analysis is altered to suit an animal species to be measured (see, for example, U.S. Pat. No. 6,391,263).
When a quaternary ammonium salt-containing lysing reagent for human blood is used to measure a canine or feline blood sample by an electric resistance detection method, the distribution of leukocyte sizes appears as a single peak, so the erythrocytes cannot be classified into a plurality of sub-groups (see, for example, WO 2002/014861, J. Anim. Clin. Med., 10(3) 129-134, 2001). This is because canine or feline leukocytes are fragile and sensitive to the quaternary ammonium salt as compared with human leukocytes.
To cope with this problem, WO 2002/014861 supra describes a method of measuring leukocytes by using the quaternary ammonium salt at a lower concentration than that for human blood (that is, the concentration of the salt in a solution measured for leukocytes is 1.5 to 4.5 g/L). It is described therein that according to this method, leukocytes in a blood sample such as a canine or feline sample can be classified into 3 sub-groups, that is, lymphocytes, “mixed cell group” composed mainly of monocytes, and granulocytes.
However, the method described in WO 2002/014861 supra cannot classify eosinophils into a sub-group by distinguishing them from other leukocytes. In examination of leukocytes, the number of eosinophils is information useful for diagnosis of allergic diseases and parasitosis. Particularly, canine and feline parasitosis is well known in the field of pet, and canine and feline allergic diseases are rapidly increasing in recent years. For diagnosis of allergic diseases and parasitosis for canine and feline, therefore, it is important that in blood examination, eosinophils be classified by distinguishing them from other leukocytes.
When the present inventors compared a result obtained by the method described in WO 2002/014861 supra with a result obtained by a visual examination, the correlation therebetween was low.